Promoter analysis of seed-specific genes has a rich history (reviewed in Goldberg et al. (1989) Cell, 56; 149-160; Thomas (1993) Plant Cell, 5; 1401-1410). This stems from the observation that no plant gene is more tightly regulated in terms of spatial expression than those encoding seed storage proteins. Many seed storage protein genes have been cloned from diverse plant species, and their promoters have been analyzed in detail (Thomas, 1993). In these experiments promoter elements, which constitute the 5xe2x80x2-upstream regulatory regions, were functionally defined by their ability to confer seed-specific expression of the bacterial xcex2-glucuronidase (GUS) reporter gene in transgenic plants (Bogue et al. (1980) Mol. Gen. Genet., 222; 49-57; Bustos et al. (1989) Plant Cell, 1; 839-853). Results of this work initiated efforts to functionally define cis-elements in the promoters of these genes that are critical for conferring seed-specific expression.
Later experiments involved construction of deletion mutants consisting of target promoters fused to the GUS-reporter gene. Analysis of these constructs in transgenic plants allowed researchers to define regions within each promoter that are critical to its overall regulation (Bustos et al. (1991) EMBO J., 10; 1469-1479; Chung (1995) Ph.D. Dissertation, Texas AandM University; Nunberg et al. (1994) Plant Cell, 6; 473-486). A general conclusion from this work is that the promoter proximal region contributes primarily to the gene""s tissue specificity with more distal regions being responsible for modulating expression levels (Thomas, 1993). In addition to this, several groups have identified and characterized specific cis-regulatory elements, in both the promoter proximal region (PPR) and more distal regions, which interact with DNA binding proteins (Bustos et al., 1989; Chung, 1996; Jordano et al. (1989) Plant Cell, 1; 855-866; Nunberg et al., 1994). The functional significance of these regulatory elements varies from gene to gene.
In some cases, cis-regulatory elements have been mapped and the trans-acting factors which confer functionality have been cloned. For example, elements that allow the wheat EM-gene to respond to the plant hormone abcisisic acid (ABA) have been defined. This work led to the identification of a DNA binding protein which mediates this response (Guiltinan et al. (1990) Science, 250; 267-271; Marcotte et al. (1989) Plant Cell, 1; 969-976). Putative ABA responsive elements have also been mapped in the sunflower helianthinin promoter HaG3-D and the carrot Dc3 promoter (Chung, 1995; Nunberg et al., 1994). Alone these elements act as positive elements in response to ABA. Regulation is restricted to the embryo, however, in the presence of each gene""s promoter proximal region (Thomas, 1993).
Despite considerable effort, the cis-regulatory elements which contribute to a promoter""s seed-specificity remain elusive (Chung, 1996; Li (1995) Ph.D. Dissertation, Texas AandM University). Recent work on the carrot Dc3 promoter proximal region has identified two bZIP genes that functionally interact with critical cis-elements (Kim et al. (1997) Plant J., 11; 1237-1251). This work has increased the understanding of seed-specific gene expression but it has also revealed that seed-specific gene regulation is complex.
In Arabidopsis thaliana, the promoters driving the expression of four members of the 2S albumin gene family have been analyzed in detail. The data indicate that each promoter is capable of conferring seed specific expression of a reporter gene in transgenic plants. Each promoter, however, confers slightly different spatial accumulation of the reporter in the developing seed. Thus, each family member contributes to the overall accumulation of the 2S albumins in the developing embryo. This is not unusual behavior for small gene families in plants (Lam et al. (1995) Plant Cell, 7; 887-898; Conceicao et al. (1994) Plant J., 5; 493-505; Sjxc3x6dahl et al. (1993) Plant Mol. Biol., 23; 1165-1176; Pang et al. (1988) Plant Mol. Biol., 11; 805-820). In such cases, each member is capable of functionally complementing the others. The expression of each member is under different regulatory control leading to unique expression patterns. This appears to be a widespread gene regulatory mechanism in plants.
There is substantial interest in identification and isolation of regulatory elements for use in manipulating expression of both native and heterologous genes in plant seeds. For example, well-defined seed specific regulatory elements are useful in manipulating fatty acid synthesis or lipid metabolism genes in plant seeds. Other important agronomic traits such as herbicide and pesticide resistance, and drought tolerance may also be altered in the plant seed by transforming plants with appropriate heterologous genes under the control of well-defined seed-specific promoters and cis regulatory elements.
The present invention provides regulatory sequences which direct seed-specific expression beginning with the early embryo and include a promoter from a seed-specific gene designated KNAT411. The subject promoters are active at a much earlier stage in embryo development than other known seed-specific promoters. The regulatory sequences may be used with any native or heterologous gene or portion thereof for expression of a corresponding gene product in a plant seed.
The present invention is directed to isolated nucleic acids comprising promoters which direct seed-specific expression beginning in the early embryo. The subject promoters hybridize under stringent hybridization conditions to a promoter isolated from Arabidopsis, designated the KNAT411 promoter, which promoter has the nucleotide sequence as set forth in SEQ ID NO:1.
The present invention is also directed to an isolated nucleic acid comprising a KNAT411 promoter which directs seed-specific expression beginning in the early embryo, which KNAT411 promoter has a restriction map as depicted in FIG. 7.
In other embodiments of the invention, the subject isolated nucleic acids comprise promoters which direct seed-specific expression beginning in the early embryo and have a sequence identity (sequence similarity) of from about 60% to about 65%, or from abut 65% to about 75%, or from about 75% to about 85% when compared to the nucleotide sequence of the KNAT411 promoter as set forth in SEQ ID NO:1. In a preferred embodiment, an isolated nucleic acid comprising a promoter which directs seed specific expression beginning early in the development of the embryo has a sequence identity (sequence similarity) of about 85% to about 90% when compared to the sequence of the KNAT411 promoter as set forth in SEQ ID NO:1. In a most preferred embodiment, an isolated nucleic acid comprising a promoter which directs seed specific expression beginning early in the development of the embryo has a sequence identity embryo has a sequence identity (sequence similarity) of about 90% or greater when compared to the sequence of the KNAT411 promoter as set forth in SEQ ID NO:1.
In a further embodiment, the present invention is directed to an isolated nucleic acid comprising a promoter which directs seed-specific expression beginning early in the development of the seed, which promoter has the nucleotide sequence as set forth in SEQ ID NO:1.
Other embodiments of the present invention include vectors comprising a subject isolated nucleic acid constituting a promoter which directs seed-specific expression beginning in the early embryo including a KNAT411 promoter or portion thereof.
In still another embodiment, the present invention is directed to cells and plants transformed with a vector comprising an isolated nucleic acid constituting a promoter which directs seed-specific expression beginning in the early embryo including a KNAT411 promoter or portion thereof, and the progeny generated from such transformed plants.
In still further embodiments, the present invention provides expression cassettes which comprise an isolated nucleic acid constituting a promoter which directs expression beginning in the early embryo of a seed including a KNAT411 promoter or portion thereof operably linked to a heterologous gene or a nucleic acid encoding a sequence complementary to the native plant gene and vectors containing such expression cassettes.
In still another embodiment, this invention provides a method for directing seed-specific expression in a plant by providing such plant with an isolated nucleic acid constituting a promoter which directs expression beginning in the early embryo including a KNAT411 promoter to effect such seed-specific expression.
The present invention also contemplates expression cassettes which comprise an isolated nucleic acid constituting a promoter which directs expression in the early embryo of a seed including a KNAT411 promoter, operably linked to a recombinase gene such as Cre or FLP (Odell et al. 1994 Plant Physiol. 106:447-458 and references therein). Such expression cassettes are useful for inducing site specific recombination in the early plant embryo. Transgenic plants comprising such expression cassettes activate a recombination system early in embryogenesis resulting in the recombination event being fixed in the germline of the plant.